MOLECULAR MECHANISMS IN THE REGULATION OF NEUROTRANSMITTER 
RECEPTOR FUNCTION 
Richard L. Huganir, Ph.D., Associate Investigator 
Recent studies in many laboratories have pro- 
vided evidence that protein phosphorylation is one 
of the major mechanisms in the control of synaptic 
transmission. Signal transduction at the postsynaptic 
membrane of chemical synapses is mediated by spe- 
cific receptors that bind the neurotransmitter and 
transduce the signal to the interior of the cell. Dr. 
Huganir and his colleagues have been investigating 
the role of protein phosphorylation of neurotrans- 
mitter receptors in the regulation of synaptic trans- 
mission. They have used the nicotinic acetylcholine 
receptor (AChR), the protorypic neurotransmitter 
receptor, as a model system. In addition, they have 
been studying the role of protein phosphorylation 
in the regulation of the major excitatory and inhibi- 
tory neurotransmitter receptors in the brain, the re- 
ceptors for glutamate and 7-aminobutyric acid. 
Characterization of Protein Phosphorylation 
of the Nicotinic Acetylcholine Receptor 
The AChR is a well-characterized neurotransmit- 
ter receptor that has served as a model system for the 
study of the structure, function, and regulation of 
neurotransmitter receptors and ion channels. The 
AChR is a pentameric complex of four types of sub- 
units in the stoichiometry of a2^yd. Studies in Dr. 
Huganir's laboratory have shown that the AChR is 
phosphorylated by cAMP-dependent protein kinase 
(PKA), protein kinase C (PKC), and a protein- 
tyrosine kinase. The phosphorylation of the recep- 
tor by cAMP-dependent protein kinase and the 
protein-tyrosine kinase has been demonstrated to 
regulate the rate of desensitization of the AChR. De- 
sensitization, a common property of most receptors, 
is a process by which a receptor is inactivated in the 
presence of its neurotransmitter. 
The functional effects of phosphorylation of the 
AChR are being studied in more detail by site- 
specific mutagenesis of the phosphorylation sites on 
the AChR subunits. All of the known phosphoryla- 
tion sites have been mutated, and the mutant AChR 
has been expressed in Xenopus oocytes and human 
embryonic kidney (HEK) cells. Expression of the 
mutant subunits produces a fully assembled recep- 
tor, though the receptor is not phosphorylated. The 
mutant AChR has been shown to be functional using 
both intracellular and single-channel recording 
techniques. The desensitization kinetics of the mu- 
tant AChR are currently being analyzed and com- 
pared with the wild-type AChR. 
Dr. Huganir's laboratory has investigated the neu- 
rotransmitters, hormones, and neuropeptides that 
regulate the level of AChR phosphorylation . A neuro- 
peptide, calcitonin gene-related peptide (CGRP), 
which is a cotransmitter with acetylcholine at cho- 
linergic synapses, has been found to regulate phos- 
phorylation of the AChR by PKA. In addition, the 
neurotransmitter that regulates PKC phosphoryla- 
tion of the AChR may be acetylcholine itself. Acetyl- 
choline has been found to regulate AChR phosphor- 
ylation in an identical manner to that of phorbol 
esters, potent activators of PKC. The acetylcholine- 
induced phosphorylation is dependent on extracel- 
lular calcium, suggesting that calcium influx 
through the AChR is crucial for the activation 
of PKC. 
The extracellular signals that regulate the tyrosine 
phosphorylation of the AChR have been elusive. 
However, studies in Dr. Huganir's laboratory funded 
by the National Institutes of Health have suggested 
that the presynaptic neuron is intimately involved in 
the activation of the protein-tyrosine kinase. In in- 
nervated rat diaphragm, the AChR is highly phos- 
phorylated on tyrosine residues. However, denerva- 
tion of the muscle leads to a progressive decrease in 
tyrosine phosphorylation. Moreover, during devel- 
opment, tyrosine phosphorylation of the AChR does 
not occur until after innervation of the muscle. Ex- 
periments using cocultures of chick myotubes and 
chick ciliary ganglion neurons have shown that in- 
nervation of the myotube in vitro stimulates tyro- 
sine phosphorylation of the AChR. These results 
strongly suggested that something from the nerve, 
either a diffusible substance or the physical contact 
of the nerve terminal itself, activates tyrosine 
phosphorylation . 
Recent studies in collaboration with Dr. Bruce 
Wallace (University of Colorado) have provided evi- 
dence that a neuronal extracellular matrix protein, 
agrin, may be the factor from neurons that regulates 
tyrosine phosphorylation of the AChR. Agrin, re- 
leased by the nerve, mediates nerve-induced aggre- 
gation of the AChR at the synapse. Treatment of 
myotubes in culture with purified agrin stimulates 
tyrosine phosphorylation of the AChR and induces 
AChR aggregation. Moreover, inhibition of the 
agrin-induced tyrosine phosphorylation by tyrosine 
kinase inhibitors eliminates the agrin-induced ag- 
gregation of the receptor. These results suggest that 
tyrosine phosphorylation may mediate agrin- 
induced AChR aggregation and formation of the 
synapse. 
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